The invention relates to radioprotectors, processes for their preparation and their use in therapy, particularly in cancer radiotherapy where they may be used to protect biological materials from radiation damage.
It is generally accepted that DNA is the crucial target in the cytotoxic effects of ionising radiation. There is considerable evidence to support the view that DNA double-stranded (ds) breaks are particularly important. The DNA damage results from both direct ionisation in the DNA molecule (direct effect) and by indirect effects mediated by the radiolysis products of water. Carbon-centred radicals on the deoxyribose moiety of DNA are thought to be the precursors of strand breaks.
The treatment of tumours with ionising radiation (hereinafter referred to as xe2x80x9ccancer radiotherapyxe2x80x9d) is used extensively in cancer therapy. The goal of such treatment is the destruction of tumour cells and inhibition of tumour cell growth presumably through DNA damage, while minimising damage to non-tumour cells and tissues. Damage to non-tumour cells often limits the effectiveness of radiotherapy of certain tumours, as exemplified by brain tumours and tumours in the abdominal cavity.
Cancer radiotherapy is a very significant public health activity. Given the incidence of cancer in the population and the international assessment that more than 50% of cancer patients benefit from inclusion of radiotherapy in their treatment, more than 10% of the population are likely to experience cancer radiotherapy in their lifetime.
The dominant consideration in prescribing radiation doses for cancer radiotherapy is the assessment of tolerance of the most radiosensitive normal tissues/organs in the treatment field. This assessment, together with the expected radiation dose required to eradicate a tumour determines whether the treatment strategy is aimed at cure or palliation. In many cases, the maximum tolerable doses are insufficient to eradicate the tumour. This dilemma is embodied in the concept of therapeutic ratio, which represents the ratio of probabilities of tumour control versus normal tissue morbidity. Approaches to improving the therapeutic ratio include:
(a) optimising the physical targeting of the radiation to the tumour;
(b) fractionation of the radiation dose; and
(c) the use of radiomodifiers.
Improving the physical delivery of radiation has had a considerable impact on the practice of radiotherapy. For example, increasing the energy of x-ray photons from several hundred kilovolts to the present-day megavoltage beams enables the zone of maximum radiation dose to be set at depths of several centimetres, whereas with the older machines the maximum dose was near the skin surface. There are a number of more sophisticated approaches to xe2x80x9ctailoringxe2x80x9d treatment beams in various stages of development and implementation. Brachytherapy, the use of implanted radioactive sources rather than external beams, is a further approach to improving the physical dose distribution.
Almost without exception, curative external beam radiotherapy involves fractionation of the radiation dose. An example of a conventional schedule would be a total of 50 Grays given in twenty-five 2 Gray fractions. Since cells have the capacity to repair radiation damage between fractions, the fractionated treatment results in much less cell-kill than a single dose of 50 Gray. However, normal cells generally have a greater repair capacity than do tumour cells, so the xe2x80x9cspacingxe2x80x9d effect of fractionation is more marked for normal tissues. In short, fractionation improves the therapeutic ratio.
Exploration of radiomodifiers such as radioprotectors and radiosensitisers has focussed on hypoxic cell sensitisers such as metranidazole and misonidazole. Radioprotectors have received much less attention than radiosensitisers at the clinical level. The nuclear era spawned considerable effort in the development of radioprotectors with more than 4000 compounds being synthesised and tested at the Walter Reed Army Institute of Research in the United States of America in the 1960""s. With the exception of a compound known as WR2727 none of the compounds have proved useful in either the military or industrial contexts (i.e., protection against total body irradiation) or for cancer radiotherapy.
It is important to note the interplay between these three approaches to improving the therapeutic ratio. A combination of improved physical targeting, fractionation and radiomodifiers could transform the intent in some radiotherapy situations from palliative to curative. For curative schedules, successful application of radiomodifiers would relax the requirement for fractionation and hence reduce overall costs of treatment, which to a large extent is proportional to the number of treatment fractions per patient.
A particularly important role for radioprotectors has emerged from the recent recognition that accelerated repopulation of tumour cells during radiotherapy can seriously compromise the effectiveness of treatment. The main consequences of this have been as follows:
(i) The development of accelerated treatment schedules to reduce the overall time of radiotherapy treatment. In such accelerated schedules, acute reactions are a particular problem, for example, acute oral mucositis in head and neck cancer patients indicate a clear need for radioprotectors.
(ii) The recognition that the interruption of radiotherapy treatment due to normal tissue reactions will reduce the probability of tumour control. Use of radioprotectors to prevent toxicity-induced treatment interruption would be clearly beneficial.
The radioprotective properties of the minor groove binding DNA ligand Hoechst 33342 were first described by Smith, P. J. and Anderson, C. O.1, who used clonogenic survival assays of irradiated cultured cells. Young, S. D. and Hill, R. P.2 reported similar effects in cultured cells, but extended their studies to in vivo experiments. They concluded that the lack of radioprotection in their in vivo experiments was due to insufficient levels of Hoechst 33342 being delivered to target cells following intravenous injection. The findings of Hill and Young underline an important requirement for effective radioprotectors, namely potency. If the radioprotector is more potent, then it is more likely to achieve the required concentrations in an in vise setting.
There is another aspect to be considered apart from potency. The concentration required for radioprotection must be non-toxic regardless of the potency of the radioprotector. If the radioprotector is delivered systemically, then this lack of toxicity requirement includes not just the cells and tissues to be protected from the radiation, but extends to the toxicity of the subject as a whole. In the case of Hoechst 33342, its toxicity limits the extent to which it is useful as a radioprotector.
There is also a substantial conceptual problem in using radioprotectors in cancer radiotherapy. In attempting to decrease the effect of radiation on normal tissues by application of radioprotectors, there is a fear that some of the radioprotector will reach the tumour, thereby compromising tumour cell kill. The existing radioprotectors, e.g. WR2721, are relatively small, diffusible molecules which do not avidly bind to tissue components and can therefore penetrate effectively through cell layers, so that they can reach the tumour via the circulation.
There is a need for radioprotectors that have limited penetration through cell layers. Such a property enables radioprotectors to be applied locally or topically to critical radiosensitive normal tissues in the vicinity of the tumour. Limited penetration restricts the extent to which the radioprotector reaches the capillary bed and is taken up into the circulation thereby reaching the tumour by systemic delivery in sufficient concentrations to confer significant radioprotection to the tumour.
The limited diffusion of DNA-binding ligands such as Hoechst 33342 through cell layers is known and has been exploited in mapping the location of cells in multi-cellular spheroids and in vivo. In addition to restricting access to the tumour by systemic uptake following local or topical application to normal tissues, there is a further potential advantage of limited penetration in the context of cancer radiotherapy. This advantage stems from the view that the vasculature, in particular the endothelial cells, are the critical targets that determine the damaging effects of radiation. Furthermore, most radioresistant cells in the tumour are those viable cells that are most distant from the capillaries. The radioresistance of these cells is due to their hypoxic state, which in turn reflects their remoteness from the capillaries.
Consequently, radioprotectors having limited diffusion, when administered intravenously, will be delivered more efficiently to critical radiosensitive cells in animal tissues, than to the subpopulation of cells in tumours (ie. hypoxic cells) which limit the effectiveness of radiotherapy generally. Thus, the use of such radioprotectors enables higher radiation doses to be used, with increased probability of killing the hypoxic cells in the tumour.
However, the potential of the combination these radiobiological features and the characteristics of DNA-binding radioprotectors can only be useful in cancer radiotherapy provided that an over-riding and necessary requirement of the radioprotectors exists, namely that the radioprotectors are sufficiently potent as to confer demonstrable radioprotection at non-toxic concentrations, when applied topically or systemically. A further practical requirement is that the extent of the limited penetration is sufficient to prevent significant systemic uptake following topical application, but not so pronounced so as to prevent sufficient concentrations from reaching the cells that determine the radiosensitivity of the tissue to be protected from the effects of ionising radiation, by topical or local application.
A requirement accordingly exists for radioprotectors which have a reduced cytotoxicity, increased radioprotective potency and a limited penetration through cell layers which can be used in cancer radiotherapy, in particular topically to protect tissues such as the skin, oral mucosa, oesophageal mucosa, rectal mucosa, vaginal mucosa and bladder epithelium and parenterally to protect organs such as the lung and brain.
According to a first aspect of the present invention there is provided a radioprotector comprising a compound of formula (I): 
wherein
X is optionally substituted aminoalkyl, optionally substituted alkylene or an interactive group;
Y and Z may be the same or different and are selected from N, O, S and C(Rxe2x80x2) wherein Rxe2x80x2 is hydrogen, optionally substituted alkyl or optionally substituted alkenyl;
{overscore (----)} is a double bond unless the attached Y or Z group is or S in which case it is a single bond;
and R1 to R11 may be the same or different and are selected from hydrogen, a sterically hindering group and an electron donating group; or any two of R1 to R11 Y, Z, NH and Rxe2x80x2 may together with the carbon atoms to which they are attached form an optionally substituted ring which may contain heteroatoms, provided that at least one of R1 to R11 is an electron donating group and that when X is NCH3, Y and Z are N and R1, R2 and R4 to R11 are hydrogen, then R3 is not OH or OCH2CH3; and salts thereof, pharmaceutically acceptable derivatives thereof, prodrugs thereof and/or tautomers thereof.
The present invention also provides the use of a compound of formula (I) defined above as a radioprotector.
The present invention further provides a compound of formula (I) defined above when used as a radioprotector.
According to a second aspect of the present invention there is provided a method for protecting a subject from radiation damage which comprises administering an effective amount of a compound of formula (I) as defined above to the subject.
According to a third aspect of the present invention there is provided a method for protecting biological materials which comprises contacting the biological material with a compound of formula (I) as defined above for a time sufficient to allow association of the compounds with DNA in the biological material.
According to a fourth aspect of the present invention there is provided a method of cancer radiotherapy which comprises administering to a subject in need of such therapy an effective amount of a compound of formula (I) as defined above and subjecting the locus of a tumour to a radiation source.
The present invention also provides the use of the compound of formula (I) defined above in protecting a subject from radiation damage, protecting biological materials or in cancer radiotherapy.
The present invention further provides the use of the compound of formula (I) defined above in the manufacture of a medicament for protecting a subject from radiation damage, protecting biological materials or in cancer radiotherapy.
The present invention still further provides a compound of formula (I) when used in the methods defined above.
Throughout this specification, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, or variations such as xe2x80x9ccomprisesxe2x80x9d or xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
The term xe2x80x9cinteractive groupxe2x80x9d is used herein in its broadest sense and refers to a group capable of forming a bond with a specific group on a target molecule such as a protein or a derivative thereof. Examples of interactive groups include N(CH2)nCOOH, N(CH2)nCO(CH2)mR, N(CH2)nxe2x80x94SH, N(CH2)nxe2x80x94NH2, CH(CH2)nCOOH, CH(CH2)nCO(CH2)mR, CH(CH2)nxe2x80x94SH and CH(CH2)nxe2x80x94NH2 wherein n is 1 to 10, m is 0 to 10 and R is optionally substituted alkyl.
The term xe2x80x9celectron donating groupxe2x80x9d is used herein in its broadest sense and relates to a substituent which confers an increase in electron density to the molecule to which it is attached. The mechanism of this electron donating effect can involve either an inductive (through bonds effect) which only operates over relatively short distances (a few inter-atomic bonds), or the xe2x80x9cresonancexe2x80x9d effect in aromatic or conjugated unsaturated bonds in aliphatic molecules. In conjugated systems, the resonance effect can be affected over extended distances. The driving force for this electron-donating propensity can be a lone electron pair on one of the atoms of the electron-donating group (eg. an amino group), or an innate low level of electronegativity (eg. an alkyl group). In the latter case, when such a group is attached to a more electrophilic system, it becomes polarised and has the effect of increasing the electron density in the group onto which it is substituted. Systematic evaluation of quantitative effects of different electron donating/electron withdrawing substituents was first reported by Hammett, who described the effect of different substituents on the acidity of analogues of benzoic acid, and led to the derivation of constants for each substituent (see review by Jaffe, Chemical Reviews, 53, 191 (1953)). The constants are described by the Hammett equation, the generalised form of which is:
logk/k0="sgr"xcfx81
where:
k and k0 are the rate constants or equilibrium constants for the substituted and parent reactants, respectively,
"sgr" is a constant characteristic of the substituent X, and
xcfx81 is also a constant characterising the particular reaction.
Table A below shows Hammett constants for a number of substituents, and some examples donating substituents, in decreasing order of electron-donor power:
(CH3)2xe2x80x94Nxe2x80x94 greater than CH3CH2Oxe2x80x94 greater than CH3xe2x80x94 greater than H greater than I greater than NO2xe2x80x94
Electron donating groups therefore include optionally substituted alkyl, optionally substituted alkebyl, NHR or NR2 and OR where R is hydrogen or optionally substituted alkyl. Preferably the electron donating group is NHR or NR2. It is postulated that the presence of at least one electron donating group increases the radioprotective activity.
The term xe2x80x9csterically hindering groupxe2x80x9d is used herein in its broadest sense to include any bulky group which stereochemically restricts, for example, the rotation or the conformation of the compound of formula (I). Sterically hindering groups are preferably groups appropriately positioned and of sufficient size to restrict the rotation about one of the dihedral bonds in the molecule (eg. piperazine-benzimidazole, benzimidazole-benzimidazole, benzimidazole-phenyl), or to otherwise prevent intramolecular stacking of the compounds of formula I. Examples of sterically hindering groups include those described above as electron donating groups which may be located, for example, adjacent to the single bonds linking the rings so as to restrict rotation. Other sterically hindering groups include optionally substituted rings which may contain heteroatoms. It is postulated that the stereochemical restriction also increases the radioprotection activity by increasing the extent of minor groove DNA-binding. This increase may be achieved by decreasing the extent of other forms of binding of the ligand to itself, to DNA (modes of binding other than minor groove binding) or to other cellular components. It is possible that some of these other forms of binding may be favoured by a coplanar conformation of the ring system of the radioprotector.
Suitable compounds of formula (I) having electron donating groups, optionally substituted rings, and/or sterically hindering groups are as follows 
wherein
R1, R2, R3 and R4 are as defined above and R is a sterically hindering group.
The term xe2x80x9calkylxe2x80x9d used either alone or in compound words such as xe2x80x9coptionally substituted alkylxe2x80x9d, xe2x80x9coptionally substituted aminoalkylxe2x80x9d or xe2x80x9coptionally substituted alkylenexe2x80x9d denotes straight chain, branched or mono- or poly-cyclic alkyl, preferably C1-30 alkyl or cycloalkyl. Examples of straight chain and branched alkyl include methyl, ethyl, proply, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, amyl, isoamyl, sec-amyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2,-trimethylpropyl, 1,1,2-trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3,-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-methyloctyl, 1-, 2-, 3-, 4-, 5-ethylheptyl, 1-, 2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1- 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-ethylnonyl, 1-, 2-, 3-, 4- or 5-propyloctyl, 1-, 2- or 3-butyloctyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4-butyloctyl, 1-2-pentylheptyl and the like. Examples of cyclic alkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl and the like.
The term xe2x80x9calkenylxe2x80x9d used either alone or in compound words such as xe2x80x9coptionally substituted alkenylxe2x80x9d denotes groups formed from straight chain, branched or mono- or poly-alkenes including ethylenically mono- or poly- unsaturated alkyl or cycloalkyl groups as defined above, preferably C2-30 alkenyl. Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl, 1-4,pentadienyl, 1,3-cyclopentadienyl, 1,3-hexadienyl, 1,4-hexadienyl, 1,3-cyclohexadienyl, 1,4-cyclohexaidenyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl, 1,3,5,7-cyclooctatetraenyl and the like.
The term xe2x80x9coptionally substituted ring which may contain heteroatomsxe2x80x9d is used herein in its broadest sense to refer to a saturated or unsaturated, homogenous or heterogeneous cyclic group, such as, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl or heterocyclyl which may contain heteroatoms selected from oxygen, nitrogen and sulphur. Examples of cycloalkyl and cycloalkenyl are described above. Suitable aryl includes single, polynuclear, conjugated and fused residues of aromatic hydrocarbons, such as, phenyl, biphenyl, terphenyl, quaterphenyl, phenoxyphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl and the like. Examples of heterocyclyl include N-containing heterocyclic groups, such as, unsaturated 3 to 6 membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, for example, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl or tetrazolyl;
saturated 3 to 6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms, such as, pyrrolidinyl, imidazolidinyl, piperidino or piperazinyl;
unsaturated condensed heterocyclic groups containing 1 to 5 nitrogen atoms, such as, indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl or tetrazolopyridazinyl;
unsaturated 3 to 6-membered heteromonocyclic group containing an oxygen atom, such as, pyranyl or furyl;
unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms, such as, thienyl;
unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, oxazolyl, isoxazolyl or oxadiazolyl;
saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as morpholinyl;
unsaturated condensed heterocyclic group containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, such as, benzoxazolyl or benzoxadiazolyl;
unsaturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as thiazolyl or thiadiazolyl;
saturated 3 to 6-membered heteromonocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, thiazolidinyl; and
unsaturated condensed heterocyclic group containing 1 to 2 sulphur atoms and 1 to 3 nitrogen atoms, such as, benzothiazolyl or benzothiadialyl.
In this specification xe2x80x9coptionally substitutedxe2x80x9d means that a group may or may not be further substituted with one or more groups selected from alkyl, alkenyl, alkynyl, aryl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, hydroxy, alkoxy, alkenyloxy, alkynyloxy, aryloxy, carboxy, benzyloxy haloalkoxy, haloalkenyloxy, haloalkynyloxy, haloaryloxy nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, azido, amino, alkylamino, alkenylamino, alkynylamino, arylamino, benzylamino, acyl, alkenylacyl, alkynylacyl, arylacyl, acylamino, acyloxy, aldehydo, alkylsulphonyl, arylsulphonyl, alkylsulphonylamino, arylsulphonylamino, alkylsulphonyloxy, arylsulphonyloxy, heterocyclyl, heterocycloxy, heterocyclylamino, haloheterocyclyl, alkylsulphenyl, arylsulphenyl, carboalkoxy, carboaryloxy, mercapto, alkylthio, arylthio, acylthio and the like.
The salts of the compound of formula (I) are preferably pharmaceutically acceptable, but it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present invention, since these are useful as intermediates in the preparation of pharmaceutically acceptable salts. Examples of pharmaceutically acceptable salts include salts of pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicyclic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
By xe2x80x9cpharmaceutically acceptable derivativexe2x80x9d is meant any pharmaceutically acceptable salt, hydrate, solvate or any other compound which, upon administration to the subject, is capable of providing (directly or indirectly) a compound of formula (I) or an active metabolite or residue thereof.
The term xe2x80x9cpro-drugxe2x80x9d is used herein in its broadest sense to include those compounds which are converted in vivo to compounds of formula (I).
The term xe2x80x9ctautomerxe2x80x9d is used herein in its broadest sense to include compounds of formula (I) which are capable of existing in a state of equilibrium between two isomeric forms. Such compounds may differ in the bond connecting two atoms or groups and the position of these atoms or groups in the compound.
The compounds of the invention may be electrically neutral or be polycations with associated anions for electrical neutrality. Suitable associated anions include sulphate, tartrate, citrate, chloride, nitrate, nitrite, phosphate, perchlorate, halosulfonate or trihalomethylsulfonate.
Preferred compounds of formula (I) are those containing N (optionally substituted alkyl)2 as the electron donating group, for example, compounds of formula (I) wherein X is NCH3, Y is N, Z is N, R3 is N(CH3)2 and R1, R2 and R4 to R11 are hydrogen (hereinafter referred to as xe2x80x9cpara dimethylamino Hoechstxe2x80x9d) or X is NCH3, Y is N, Z is N, R3is N(CH3)2, R1 is CH3 and R2 and R4 to R11 are hydrogen (hereinafter referred to as xe2x80x9cortho methyl para dimethylamino Hoechstxe2x80x9d).
According to another aspect of the present invention there is provided a radioprotector comprising para dimethylamino Hoechst.
The present invention also provides a method of protecting a subject from radiation damage which comprises administering an effective amount of para dimethylamino Hoechst to the subject.
The present invention further provides a method for protecting biological materials which comprises contacting the biological material with para dimethylamino Hoechst for a time sufficient to allow the association of this compound with the DNA in the biological material.
The present invention still further provides a method of cancer radiotherapy which comprises administering to a subject in need of such therapy an effective amount of a para dimethylamino Hoechst and subjecting the locus of the tumour to a radiation source.
Some of the compounds of formula (I) are novel per se. Thus, the present invention also provides a compound of formula (Ia) which is a compound of formula (I) as defined above with the provisos that:
(i) at least one of R1 to R11 is an electron donating group, and
(ii) when X is NCH3, Y and Z are N and R1, R2 and R4 to R11 are hydrogen, then R3 is not OH, OCH3CH3, N(CH3)2, CH3, alkyl, phenyl or Ophenyl; and
(iii) when X is NCH3, Y and Z are N and R3 to R11 are hydrogen, neither R1 nor R2 is OH or NMe2; and
(iv) when X is NCH3, Y and Z are N and R1and R3 to R11 are hydrogen, R2 is not CH3, Oalkyl or NH2; and
(v) when X is CH2, Y and Z are N and R1, R2 and R4 to R11 are hydrogen, R3 is not NMe2; and
(vi) when X is CH2, Y and Z are N and R1 and R4 to R11 are hydrogen, R2 is not NH2; and
(vii) when X is N, CH3, Y and/or Z is/are O, and R1, R2 and R4 to R11 are Hydrogen, then R3 is not OH; and salts thereof, pharmaceutically acceptable derivatives thereof, pro-drugs thereof and/or tautomers thereof.
According to another aspect of the present invention there is provided a process for the preparation of a compound of formula (Ia) in which Y and Z are selected from O, S and N which comprises either:
(A)(i) coupling a compound of formula (II): 
xe2x80x83wherein X, R9, R10 and R11 are as defined above, Y is O, S or N and n is 1 or 2 with (a), when Y is N, a compound of formula (III): 
xe2x80x83wherein Z is N, R6, R7, R8 and n are as defined above, R12 is halogen, R13 is alkyl and R14 is oxygen when Z is N or a suitable protecting group, such as an acetyl group, when Y is O or S, or with (b), when Y is N, O or S, a compound of formula (IV): 
xe2x80x83wherein R6, R7, R8 and n are as defined above and R14 is oxygen; to form a compound of formula (V): 
(ii) reducing or deprotecting the compound of formula (V) to form a compound of formula (VI): 
xe2x80x83wherein X, Y, Z, R7, R8, R9, R10, R11 and n are as defined above; and
(iii) coupling the compound of formula (VI) with either a compound of formula (VII): 
xe2x80x83wherein R1, R2, R3, R4, R5, R12 and R13 are as defined above a compound of formula (VIII): 
xe2x80x83wherein R1, R2, R3, R4, R5, R12 and R13 are as defined above; or
(B)(i) coupling a compound of formula (IX): 
xe2x80x83wherein R6, R7, R8 and n are as defined above with a compound of formula (VIII),
xe2x80x83wherein R1, R2, R3, R4 and R5 are as defined above to form a compound of formula (X): 
xe2x80x83wherein R1, R2, R3, R4, R5, R6 and R8 are as defined above;
(ii) converting the compound of formula (X) into a compound of formula (XI): 
xe2x80x83wherein Z, R1, R2, R3, R4, R5, R6, R7, R8, R12 and R13 are as defined above; or
(iii) converting the compound of formula (X) into a compound of formula (XII): 
(iii) coupling the compound of formula (XI) with a compound of formula (XIII) where Y is N or coupling a compound of formula (XII) with a compound of formula (XIII) where Y is O or S: 
xe2x80x83wherein X, R9, R10, R11 and n are as defined above.
In another aspect of the present invention there is provided a process for the preparation of a compound of formula (Ia) in which one or both of Y and Z is/are C(Rxe2x80x2) which comprises:
A(i) coupling a compound of formula (XIV): 
xe2x80x83where Rxe2x80x2, R6, R7, R8 and R13 are as defined above with a compound of formula (XV): 
xe2x80x83where R1, R2, R3, R4 and R5 are as defined above and R15 is a leaving group, such as chlorine, to form a compound of formula (XVI): 
(ii) cyclising the compound of formula (XVI) to form a compound of formula (XVII): 
(iii) coupling the compound of formula (XVII) with a compound of formula (XVIII): 
xe2x80x83where R9, R10, R11, R13 and Rxe2x80x2 are as defined above and Rxe2x80x3 is a nitrogen protecting group such as a 2-trimethylsilylethoxymethyl (SEM) group in the presence of a Pd catalyst followed by deprotection to form a compound of formula (Ia) where Y is C(Rxe2x80x2) and Z is C(Rxe2x80x2);
xe2x80x83or (iv) formylating the compound of formula (XVII) to form a compound of formula (XIX): 
(v) coupling the compound of formula (XIX) with a compound of formula (II) where Y is N to form a compound of formula (Ia) where Y is N and Z is C(Rxe2x80x2);
or B(i) coupling a compound of formula (XVII) with a compound of formula (XX): 
xe2x80x83in the present of a Pd catalyst followed by deprotection to form a compound of formula (Ia) where Y is C(Rxe2x80x2);
or C(i) coupling a compound of formula (XXI): 
xe2x80x83with a compound of formula (XVII) in the presence of a Pd catalyst to form a compound of formula (Ia) where Z is C(Rxe2x80x2)
Preferred compounds of formula (Ia) are those in which, subject to the above provisos, X is aminoalkyl,
Y and Z are selected from N and C(Rxe2x80x2) where Rxe2x80x2 is hydrogen,
R3 is an electron donating group,
R1, R5, R7 and R10 are selected from Hydrogen and a sterically hindering group, with the proviso that at least one is a sterically hindering group,
and R2, R4, R6, R8, R9 and R11 are hydrogen.
More preferred compounds of formula (Ia) are those in which, subject to the above provisos,
X is NCH3,
Y and Z are selected from N or C(Rxe2x80x2) where Rxe2x80x2 is hydrogen,
R3 is N(R)2 or NHR,
R1, R5, R9 and R10 are selected from hydrogen, optionally substituted alkyl, optionally substituted alkenyl and OR where R is alkyl with the proviso that at least one is other than hydrogen, and
R2, R4, R6, R8, R9 and R11 are hydrogen.
The most preferred compound of formula (Ia) is orthomethyl para dimethylamino Hoechst as defined above.
According to another aspect of the present invention there is provided a radioprotector comprising ortho methyl para dimethylamino Hoechst.
The present invention also provides a method of protecting a subject from radiation damage which comprises administering an effective amount of ortho methyl para dimethylamino Hoechst to the subject.
The present invention further provides a method for protecting biological materials which comprises contacting the biological material with ortho methyl para dimethylamino Hoechst for a time sufficient to allow the association of this compound with the DNA in the biological material.
The present invention still further provides a method of cancer radiotherapy which comprises administering to a subject in need of such therapy an effective amount of a ortho methyl para dimethylamino Hoechst and subjecting the locus of the tumour to a radiation source.
The subject which is protected from radiation damage may be a human or an animal such as a domestic or wild animal, particularly an animal of economic importance.
The radiation damage may result from exposure to a radiation source, such as, ionising radiation. The term xe2x80x9cionising radiationxe2x80x9d as used herein refers to photons having enough energy to ionise a bond, such as, xcex1, xcex2 and xcex3 rays from radioactive nuclei and x-rays.
The term xe2x80x9cbiological materialxe2x80x9d is used herein in its broadest sense and includes any composition of matter which comprises at least one biotechnologically-derived component. Biological material contemplated by the present invention includes proteins and other proteinaceous material including extracts of or including proteins and chemically modified proteins or extracts thereof; tissue fluids, tissue extracts or organs; animal, plant or microbiological tissue, fluid or extracts including products therefrom; biologically derived non-proteinaceous material such as, but not limited to, lipids, carbohydrates, hormones and vitamins including extracts and derivatives thereof; recombinant products including genetic material such as chromosomal material, genomic DNA, cDNA, mRNA, tRNA, ribosomes and nuclear material; and whole animal, plant or microbiological cells or extracts thereof.
The term xe2x80x9ccancer radiotherapyxe2x80x9d is used herein in its broadest sense and includes radiotherapy involving tumours which may be either benign or malignant.
The term xe2x80x9cDose Modification Factorxe2x80x9d (DMF) as used herein refers to the ratio of the radiation dose required to produce a given effect in the presence of protector, to that requires to produce the equivalent effect in the absence of protector.
The present invention also extends to a radioprotective composition which comprises a compound of formula (I) or (Ia) as defined above in association with a pharmaceutically or veterinarily acceptable carrier.
The compounds of the invention may be advantageously used in therapy in combination with other medicaments, such as, chemotherapeutic agents, for example, radiomimetic agents which are cytotoxic agents that damage DNA in such a way that the lesions produced in DNA are similar to those resulting from ionising radiation. Examples of radiomimetic agents which cause DNA stand breaks include bleomycin, doxorubicin, adriamycin, 5FU, neocarcinostatin, alkylating agents and other agents that produce DNA adducts. It is anticipated that the radioprotectors of the present invention will protect DNA from damage by some of these agents, in the same way as they protect against the effects of ionising radiation. In clinical applications, it is unlikely that the radioprotector would be administered systemically together with the chemotherapeutic agent, since this could compromise the action of this agent on the tumour. However, there are circumstances where topical application to problem tissues could be advantageous. For example, oral mucositis is a problem side-effect for cytotoxic agents, such as, doxo rubicin and administration of the present radioprotector as a mouth-wash before administration of the chemotherapeutic agent could ameliorate this side-effect without compromising the action of this agent on a tumour not located in the oral cavity. Similarly, the gastrointestinal tract could be protected by oral administration, the lungs by aerosol inhalation or the bladder by intravesical delivery, for example, via a catheter of the radioprotector. Hence a preferred method in accordance with the present invention utilises the compound of formula (I) or (Ia) in conjunction with another medicament, such as, a radiomimetic agent.
The compounds of the invention may be conjugated to agents, for example, via the interactive group, which will specifically deliver them to a desired tumour site. Suitable agents may include antibodies or proteins, such as, growth factors, for example, haemopoietic growth factor which will enable preferential radioprotection of haemopoietic stem cells to occur in the context of total body irradiation and bone marrow transplantation.
There is also an ex vivo application of the conjugates of the compounds of the invention in the context of bone marrow transplantation. Bone marrow transplantation generally involves obtaining and storing bone marrow samples from a subject in anticipation of a deterioration of their condition. A rather drastic form of chemotherapy (i.e. a high dose) is then administered. This chemotherapy is such that it would normally be lethal due to the destruction of normal stem cells, but the subject is rescued by the administration of their own haemopoietic stem cells. The problem with this procedure is that the initial sample of stem cells is likely to be contaminated with tumour cells and various procedures are used therefore to purge the bone marrow preparations of the tumour cells. Radioprotectors conjugated to a haemopoietic growth factor could be used in this context by being added to a suspension of bone marrow cells. The suspension could then be irradiated in the expectation that the normal bone marrow cells, but not the tumour cells, would be preferentially protected from the cell-killing effects of the radiation.
The compound of formula (I) or (Ia) hereinafter referred to as the xe2x80x9cactive ingredientxe2x80x9d may be administered for therapy by any suitable route, including oral, rectal, nasal, topical (including buccal and sublingual), vaginal, intravesical and parenteral (including subcutaneous, intramuscular, intravenous, intrasternal and intradermal). Preferably, administration will be by the rectal, topical, vaginal or parenteral route, however it will be appreciated that the preferred route will vary with the condition and age of the subject, the tissue/tumour being treated, its location within the subject and the judgement of the physician or veterinarian. The compound of formula (I) or (Ia) may be administered directly into tissues surrounding or proximal to tumours to be irradiated.
The compositions of the present invention comprise at least one compound of formula (I) or (Ia), together with one or more pharmaceutically acceptable carriers, diluents adjuvants and/or excipients and optionally other medicaments. Each carrier, diluent, adjuvant and/or excipient must be pharmaceutically xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the other ingredients of the composition and not injurious to the subject. Compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, intravesical or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The compositions may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers, diluents, adjuvants and/or excipients or finely divided solid carriers or both, and then if necessary shaping the product.
Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
A tablet may be made by compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (e.g inert diluent, preservative disintegrant (e.g. sodium starch glycollate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose) surface-active or dispersing agent. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with an enteric coating, to provide release in parts of the gut other than the stomach.
Compositions suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth gum; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia gum; and mouthwashes or sprays comprising the active ingredient in a suitable liquid carrier.
For topical application to the skin, the active ingredient may be in the form of a cream, ointment, jelly, solution or suspension.
For topical application to the eye, the active ingredient may be in the form of a solution or suspension in a suitable sterile aqueous or non-aqueous vehicle. Additives, for instance buffers, preservatives including bactericidal and fungicidal agents, such as phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorohexidine and thickening agents such as hypromellose may also be included.
Compositions for rectal administration may be presented as a suppository with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the active ingredient. Such excipients include cocoa butter or a salicylate.
Nasal compositions may be presented topically as nose drops or sprays or systemically in a form suitable for absorption through the nasal mucosa and/or the alveolar cells in the lungs.
Compositions suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
Compositions suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended subject; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The compositions may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
Preferred unit dosage compositions are those containing a daily dose or unit, daily sub-dose, as hereinabove described, or an appropriate fraction thereof, of an active ingredient.
The compound of formula (I) or (Ia) may also be presented for use in the form of veterinary compositions, which may be prepared, for example, by methods that are conventional in the art. Examples of such veterinary compositions include those adapted for:
(a) oral administration, external application, for example drenches (e.g. aqueous or non-aqueous solutions or suspensions); tablets or boluses; powders, granules or pellets for admixture with feed stuffs; pastes for application to the tongue;
(b) parenteral administration for example by subcutaneous, intramuscular or intravenous injection, e.g. as a sterile solution or suspension; or (when appropriate) by intramammary injection where a suspension or solution is introduced into the udder via the teat;
(c) topical application, e.g. as a cream, ointment or spray applied to the skin; or
(d) intravaginally, e.g. as a pessary, cream or foam.
It should be understood that in addition to the ingredients particularly mentioned above, the compositions of this invention may include other agents conventional in the art having regard to the type of composition in question, for example, those suitable for oral administration may include such further agents as binders, sweeteners, thickeners, flavouring agents, disintegrating agents, coating agents, preservatives, lubricants and/or time delay agents.
Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginc acid or agar. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, steric acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
The primary application of the radioprotector of the present invention is in cancer radiotherapy. Many of the normal tissues which are a problem in radiotherapy such as the skin, oral mucosa, oesophageal mucosa, rectal mucosa, vaginal mucosa and bladder epithelium can be topically protected by the radioprotectors of the present invention.
There are two distinct settings for such topical radioprotectors. Firstly, there is potential to decrease the distressing acute reactions that often occur in these tissues. Although these acute reactions can be transient, their amelioration would obviously be of benefit to a subject. A different setting is the situation where acute reactions limit the dose of radiation that can be delivered to the tumour. An example is in the accelerated fractionation regime, in which acute reactions can be dose-limiting. Thus, the application of radioprotectors could enable the use of higher radiation doses, and hence increased prospects for cure.
Aside from topical application, the pharmaco-distribution properties of the radioprotectors of the present invention offer other potential ways of achieving an improved therapeutic ratio. Examples include tumours in the brain and lung.
In the case of the brain, endothelial cells are thought to be an important radiosensitive target in terms of the detrimental effects of radiation on normal brain tissue. The administration of the radioprotector of the present invention would protect the important endothelial cells in the normal brain. The corresponding cells in the tumour would also be protected, but these cells are well oxygenated and are therefore are the most radiosensitive cells in the tumour. The more distant cells in the tumour which are hypoxic would therefore be out of reach of the radioprotector. This means that the normal endothelial cells and oxic (radiosensitive) cells of the tumour would be protected equally. This radioprotection would then enable a higher dose of irradiation to be used which would increase the chance of killing the hypoxic cells in the tumour. The fact that the endothelial cells of both the tumour and normal tissue are effected equally has no impact on the therapeutic ratio. An increase in the therapeutic ratio occurs because of the increase in kill of hypoxic tumour cells, without any debt in terms of normal tissue damage.
In the case of tumours in the lung, the radioprotector of the present invention would be delivered to alveolar cells, Although the endothelial cells of the lung tumour may also be protected, the more distant cells in the tumour would not. Moreover, the circulation of some lung tumours is provided not by the pulmonary artery but from the bronchial circulation, which will not be accessed until the next pass of the radioprotector in the circulation and hence exposed to lower concentrations.
The targeting of radioprotectors may also achieve improved therapeutic ratios in radiotherapy. A suitable example is the conjugation of the radioprotector of the present invention to haemopoietic growth factor to achieve preferential radioprotection of haemopoietic stem cells in the context of total body irradiation and bone marrow transplantation.
Outside the context of cancer radiotherapy, the radioprotectors of the present invention could be used prophylactly in high risk radiation situations. For example, the haemopoietic growth factor conjugate described above could be administered for this purpose.
The invention will now be described with reference to the following Examples. These Examples are not to be construed as limiting the invention in any way.